Chemical modifications of proteins which induce new receptor specificities and therefore elicit new effects in cells

ABSTRACT

A new reagent effective in inhibiting cholesterol synthesis 75% in human fibroblasts derived from patients suffering from the disease familial hypercholestrolemia is mannose-6-phosphate-low density lipoprotein and is effective in tissue culture test systems at 100 μg/ml after a ten-hour exposure. The broad purpose of this invention is to modify the receptor specificity of a protein so that it will enter cells which were previously impermeable and exert new effects or reverse a pathological condition. Toxins may also be modified in this manner producing cell type specific and tumor suppressive reagents which are effective in a dose range of 0.3-3 μg. The object here is to use the reagent to selectively kill one cell type which is exerting a pathological effect without affecting normal cells. Among others to which this invention is applicable are Man6P-low density lipoprotein, Man6P-ricin, Man6P-Modeccin, anti Thy 1.2 monoclonal antibody-ricin and anti Thy 1.1 monoclonal antibody-ricin.

This invention relates to a new reagent effective in inhibitingcholesterol synthesis 75% in human fibroblasts derived from patientssuffering from the disease familial hypercholestrolemia, is amannose-6-phosphate-low density lipoprotein and is effective in tissueculture test systems at 100 μg/ml after a ten-hour exposure. The broadpurpose of this invention is to modify the receptor specificity of aprotein so that it will enter cells which were previously impermeableand exert new effects or reverse a pathological condition. Toxins mayalso be modified in this manner producing cell type specific and tumorsuppressive reagents which are effective in a dose range of 0.3-3 μg.The object here is to use the reagent to selectively kill one cell typewhich is exerting a pathological effect without affecting normal cells.Among others to which this invention is applicable are Man6P-low densitylipoprotein, Man6P-ricin, Man6P-Modeccin, anti Thy 1.2 monoclonalantibody-ricin and anti Thy 1.1 monoclonal antibody-ricin (Thy are themurine thymic adult differentiation antigens).

The purpose of this invention is to modify the receptor specificity of apotent toxin by coupling it with a monoclonal antibody directed at aspecific tumor or differentiation antigen. The object generally is touse this reagent to selectively kill human tumor cells without affectingnormal cells.

A number of attempts have been made to develop tumor specific cytotoxicreagents by coupling antitumor antibodies to toxins. Early studiesfailed to show large selectivity between tumor cells and normal cellsbecause (1) toxin binding to normal cells via toxin B chain was notblocked and (2) polyclonal antibodies raised in xenogenic animals havebroad specificity and react with normal cells (Science, 169:68-70, 1970;J. Natl. Cancer Inst., 55:473-477, 1975; Nature, 271:752-755, 1978).

These antibody toxin conjugates use the entire toxin since it is shownthat the toxin B chain contains a necessary entry function in additionto its usual binding function. The toxin ricin is bound to normal cellswith lactose. In animal studies antibody-ricin conjugates are givenintravenously in hyperosmotic lactose, sufficient to raise serum lactoseto 20-30 mM. The entry function on the toxin B chain is at anintracellular site not accessible to lactose. Therefore, the entryfunction is maintained and the antibody toxin conjugate in the presenceof lactose has the same toxicity as ricin alone toward the target cell.To insure a high degree of tumor specific selectivity, antibodies are ofmonoclonal origin.

As to tumor suppressant composition used i.v. at Day 1, it has beenfound that an amount of 1-3 μg of anti Thy monoclonal antibody-ricin isa preferred dosage.

USE STATEMENT

The first purpose of this invention is to modify the receptorspecificity of a physiologic protein with the object that the proteinmay gain entrance to cells which have lost their normalreceptor-mediated uptake through a disease process. The new receptorentry route is chosen so that the protein exerts its normal physiologiceffect through the altered receptor-uptake process. Thusmannose-6-phosphate-low density lipoprotein enters familialhypercholesterolemic fibroblasts through the mannose-6-phosphatereceptor and down regulation of the rate limiting enzyme in cholesterolsynthesis is achieved. This, in turn, through feedback mechanisms, maylower the elevated levels of low density lipoprotein in patientssuffering from familial hypercholesterolemia.

The reagent of this invention is effective in inhibiting cholesterolsynthesis by 75% in human fibroblasts derived from patients sufferingfrom familial hypercholesterolemia, Man6P-low density lipoprotein, andis effective in tissue culture tests at 100 μg/ml.

The second purpose of this invention is to modify the receptorsepcificity of a toxin with the objective that the toxin will now bind,enter and kill a specific population of cells while leaving other cellsunaffected. Thus, mannose-6-phosphate ricin in the presence of lactoseselectively kills human fibroblasts while other cell types areunaffected. Monoclonal antibody toxin hybrids behave in a similarfashion.

This invention is a test or kit with some human useful linkage whichutilizes an antibody toxin conjugate where the entire toxin is utilizedand it is shown that the toxin B chain contains a necessary entryfunction in addition to its usual binding function. In animal studiesantibody ricin conjugates are given intravenously in hyperosmoticlactose sufficient to raise the serum lactose to 20-30 mM.

PRIOR ART STATEMENT

Low density lipoprotein has been modified by incorporation of cationicgroups and enters familial hypercholesterolemic fibroblasts and downregulates the rate limiting enzyme in cholesterol synthesis. [Proc.Natl. Acad. Sci. USA, 73:3178 (1976)]. Since this modification allowslow density lipoprotein to bind to many receptors on many cell types, itdoes not constitute a cell type specific reagent and therefore haslimited potential both conceptually and practically. Previous to thepresent construction of mannose-6-phosphate-low density lipoproteinthere was no reported method for efficiently introducing functionalproteins into specific cells.

The following articles describe the introduction of proteins into cellvia alternate receptors. However, the internalized proteins did notaffect the cell and the aim was not to achieve a functional proteinoperating inside the cell.

Biochem. Biophys. Res. Commun., 45:622-628, 1971.

J. Biol. Chem., 253:6107-6110, 1978.

The following articles were attempts to create cell type specific toxinsby coupling toxin A chains with hormones. Toxicity when present was toolow to be practical.

J. Biol. Chem., 252:1505-1514, 1977.

J. Biol. Chem., 252:1515-1522, 1977.

J. Biol. Chem., 254:1028-1032, 1979.

The following is the first report of the use of lactose to block thericin binding site of a hybrid toxin and the first report of intravenousinfusions of lactose protecting mice from ricin toxicity.

J. Exp. Med., 143:1464-1474, 1976.

Brit. J. Cancer, 34:418-425, 1976.

In the case of M6P-ricin and anti Thy 1.2 ricin hybrids, which containthe ricin B chain, these hybrids require the presence of lactose toblock the ricin B chain binding to achieve cell type specificity. Thislimits the presently achievable selectivity between cell types tobetween 30- and 700-fold. Naturally occurring toxins which utilizereceptor mediated protein transport systems can exhibit cell typeselectivities up to 10,000-fold. This degree of selectivity could inprinciple be also reached by antibody-toxin hybrids of the properconstruction. The currently available selectivity, however, is more thanample for the use of hybrids as selective agents for the isolation ofreceptor minus mutant cell lines. It may be possible to efficientlyselect for variants that lack any cell surface components toward whichan antibody can be raised. This approach avoids utilization ofcomplement dependent cell lysis.

The use of monoclonal antibodies as the cell recognition moiety of toxinhybrids greatly expands the possible uses of antibody-toxin hybrids.Several cell-type specific and tumor specific or tumor associatedmonoclonal antibodies have been produced. Hybrids of ricin with theseantibodies would kill the antigen bearing cells selectively. There isconsiderable scientific and pharmacologic potential for these potentmonoclonal antibody-ricin hybrids as cell type and tumor specifictoxins.

AUTOIMMUNE DISEASES

Ricin coupled to an antigen will bind, enter and kill the specific B andT cell clones which make and regulate antibody directed toward thisantigen. This will constitute a specific cure for autoimmune diseasessuch as myesthemia gravis, lupus erythematosus, rheumatoid arthritis,etc. Where the antigen in question is known and can be purified such asthe acetylcholine receptor for myesthemia gravis, it only remains tocouple the antigen to ricin and then to administer intravenously to thepatient in the presence of hyperosmotic lactose sufficient to raiseblood lactose to 20-30 mM. In autoimmune diseases where the antigen isunknown, such as rheumatoid arthritis, the antigen must be identifiedand then purified.

In retrospect, the first purpose of this invention is to modify thereceptor specificity of a physiologic protein with the object that theprotein may gain entrance to cells which have lost their normalreceptor-mediated uptake through a disease process. The new receptorentry route is chosen so that the protein exerts its normal physiologiceffect through the altered receptor-uptake process. Thus,mannose-6-phosphate-low density lipoprotein enters familialhypercholesterolemic fibroblasts through the mannose-6-phosphatereceptor and down regulation of the rate limiting enzyme in cholesterolsynthesis is achieved. This, in turn, through feedback mechanisms, maylower the elevated levels of low densitty lipoprotein in patientssuffering from familial hypercholesterolemia.

The second purpose of this invention is to modify the receptorrspecificity of a toxin with the objective that the toxin will now bind,enter and kill a specific population of cells while leaving other cellsunaffected. Thus, mannose-6-phosphate ricin in the presence of lactoseselectively kills human fibroblasts while other cell types areunaffected. Anti Thy 1.2-ricin also specifically kills cells carryingthe Thy 1.2 antigen while leaving other cells unaffected. This reagentis active against tumors carrying Thy 1.2.

The tumor suppressive composition is active against lymphoma consistingof an injection of hybrid protein anti Thy 1.2 monoclonal antibody-ricinand hyperosmotic lactose. The inoculation i.v. of murine tissues in vivoby lymphoma is made at -20 to -25 days and the tumor suppressantcomposition is used i.v. at Day 1 in an amount of 1-3 μg of anti Thymonoclonal antibody-ricin together with sufficient hyperosmotic lactoseto raise the lactose level to 20-30 mM. The broad purpose of thisinvention is to modify the receptor specificity of a potent toxin suchas ricin by coupling it with a monoclonal antibody directed at aspecific tumor or differentiation antigen. The object here is to use thereagent to selectively kill tumor cells without affecting normal cells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the toxicity of the Man6P-Modeccin via the galactoseroute of entry ( -- ), the Man6P route ( -- ) and a non-specific route (-- ).

FIG. 2 demonstrates the effect of anti Thy 1.1 monoclonal antibody-ricinon the inhibition of protein synthesis on AKR-SL2 and EL-4 cells.Protein synthesis is inhibited in AKR-SL2 cells which carry the Thy 1.1antigen. No effect is seen in this concentration range on EL-4 cellswhich lack the Thy 1.1 antigen. This demonstrates the cell typespecificity of this reagent.

EXAMPLE 1 Preparation of a New Toxic Hybrid: Man6P-Modeccin

Man6P-Modeccin was prepared by covalently coupling the toxin Modeccin toω-(6-phospho)-pentamannose. The incubation mixture consisted of: 100 μlModeccin (3 mg/ml), 100 μl ω-(6-phospho)-pentamannose (pH 8.5, 400mg/ml), 17 ul Bicine buffer (1-N,N-bis-hydroxy-2-ethylglycine;Calbiochem) (1.5 M, pH 9.0) and 12.5 μl [³ H]-NaCNBH₃. After 40 hours at37° C. the mixture was passed over a column of Sephadex G-25 Superfine(0.5×25 cm) and eluted with Dulbecco's phosphate buffered saline (6.7 mMNa₂ HPO₄, 15 mM NaCl) (pH 7.4). Incorporation of tritium label revealed7 P(Man)₅ residues incorporated per mole of protein.

Toxicity of the Man6P-Modeccin preparation was assayed in the absenceand presence of lactose and in the presence of lactose plus Man6P. Theaccompanying figure demonstrates the toxicity via the gal route of entry( -- ), the Man6P route ( -- ) and a non-specific route ( -- ). This isa clear demonstration of an alternate route of toxin entry via the Man6Preceptor.

EXAMPLE 2

In a manner similar to the preparation of Man6P-Modeccin in Example 1,Man6P-ricin and Man6P-low density lipoprotein were prepared.

We claim:
 1. A cell type specific cytotoxic reagent compositionconsisting of mannose-6-phosphate-Modeccin, a hybrid prepared fromModeccin.